Co-stimulation of the fc receptor and interleukin-12 receptor on human natural killer cells leads to increased expression of cd25.

Natural killer (NK) cells serve a critical role in the immune response against microbes and developing tumors. We have demonstrated that NK cells produce stimulatory cytokines (e.g., IFN-γ) in response to potent stimulation via immobilized IgG (to engage Fc receptors) and interleukin (IL)-12. CD25 is a component of the high-affinity IL-2R, which promotes NK cell activation in response to low doses of IL-2 such as those released by activated T cells. We hypothesized that stimulation of NK cells via IgG and IL-12 would enhance CD25 expression and promote NK cell anti-tumor activity in response to low-dose IL-2. It was confirmed that this dual stimulation strategy significantly enhanced NK cell CD25 expression compared to unstimulated cells or cells treated with IgG or IL-12 alone. Dual stimulated NK cells also were more responsive to low-dose IL-2. Dual stimulated NK cells subsequently treated with low-dose IL-2 (10 pg/mL) displayed enhanced intracellular signaling as indicated by increased pSTAT5 levels. IFN-γ production and cytotoxicity against K562 cells by NK cells stimulated with low-dose IL-2 was comparable to that of cells treated with high-dose IL-2 (10 ng/mL). Importantly, cells isolated from head and neck cancer patients receiving the mAb cetuximab and IL-12 on a clinical trial displayed increased CD25 expression following combination therapy compared to baseline. Altogether, these findings suggest that FcR and IL-12R co-stimulation induces expression of the high-affinity IL-2R and promotes NK cell anti-tumor activity.

MICA-Expressing Monocytes Enhance Natural Killer Cell Fc Receptor-Mediated Antitumor Functions.

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA-NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell-monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778-89. ©2017 AACR.

Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor.

Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+ cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cells in vivo can stimulate the increased expression of IL-21R on NK cells.

Gene expression profiling of the human natural killer cell response to Fc receptor activation: unique enhancement in the presence of interleukin-12.

BACKGROUND: Traditionally, the CD56(dim)CD16(+) subset of Natural Killer (NK) cells has been thought to mediate cellular cytotoxicity with modest cytokine secretion capacity. However, studies have suggested that this subset may exert a more diverse array of immunological functions. There exists a lack of well-developed functional models to describe the behavior of activated NK cells, and the interactions between signaling pathways that facilitate effector functions are not well understood. In the present study, a combination of genome-wide microarray analyses and systems-level bioinformatics approaches were utilized to elucidate the transcriptional landscape of NK cells activated via interactions with antibody-coated targets in the presence of interleukin-12 (IL-12). METHODS: We conducted differential gene expression analysis of CD56(dim)CD16(+) NK cells following FcR stimulation in the presence or absence of IL-12. Next, we functionally characterized gene sets according to patterns of gene expression and validated representative genes using RT-PCR. IPA was utilized for biological pathway analysis, and an enriched network of interacting genes was generated using GeneMANIA. Furthermore, PAJEK and the HITS algorithm were employed to identify important genes in the network according to betweeness centrality, hub, and authority node metrics. RESULTS: Analyses revealed that CD56(dim)CD16(+) NK cells co-stimulated via the Fc receptor (FcR) and IL-12R led to the expression of a unique set of genes, including genes encoding cytotoxicity receptors, apoptotic proteins, intracellular signaling molecules, and cytokines that may mediate enhanced cytotoxicity and interactions with other immune cells within inflammatory tissues. Network analyses identified a novel set of connected key players, BATF, IRF4, TBX21, and IFNG, within an integrated network composed of differentially expressed genes in NK cells stimulated by various conditions (immobilized IgG, IL-12, or the combination of IgG and IL-12). CONCLUSIONS: These results are the first to address the global mechanisms by which NK cells mediate their biological functions when encountering antibody-coated targets within inflammatory sites. Moreover, this study has identified a set of high-priority targets for subsequent investigation into strategies to combat cancer by enhancing the anti-tumor activity of CD56(dim)CD16(+) NK cells.

ß-Adrenergic receptor mediated increases in activation and function of natural killer cells following repeated social disruption.

Natural killer (NK) cells are specialized innate lymphocytes important in the early defense against tumor and virus bearing cells. Many factors influence the immune system's effectiveness against pathogens, including stress. Social disruption (SDR) "primes" macrophages/monocytes and dendritic cells thereby enhancing their anti-microbial function. What remains unclear is whether similar responses are evident in NK cells. Current studies investigated the cellular distribution and activation/inhibitory phenotypes of NK cells in the spleen, lung, and blood of C57BL/6 male mice following SDR. Furthermore, cytolytic activity and anti-viral cytokine production of splenic NK cells were determined. Lastly, ß-adrenergic receptor (ß-AR) signaling was investigated to determine possible mechanisms behind the SDR-induced NK cell alterations. Results indicated NK cells from SDR mice have increased expression of CD16 and CD69 and reduced NKG2a and Ly49a expression on splenic CD3-/DX5+ NK cells indicative of an activated phenotype, both immediately and 14h post-SDR. Administration of propranolol (10mg/kg; non-selective ß-adrenergic receptor antagonist) was shown to block these "priming" effects at the 14h time-point. In the lung, SDR had similar effects on activation and inhibitory receptors 14h post-SDR, however no alterations were evident in the blood besides increased NK cells directly after SDR. Additionally, splenic NK cells from SDR mice had increased CD107a surface expression, cytolytic activity, and IFN-γ production was increased upon costimulation with IgG and IL-2 ex vivo. Collectively, these data suggest that social stress "primes" NK cells in the spleen and lung to be more proficient in their cytolytic and anti-viral/tumor effecter functions through ß-adrenergic receptor dependent signaling.

Cetuximab therapy in head and neck cancer: immune modulation with interleukin-12 and other natural killer cell-activating cytokines.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Greater than 90% of SCCHN of the oropharynx overexpress the epidermal growth factor receptor (EGFR or HER1). Cetuximab (Erbitux-TM) is a humanized anti-HER1 monoclonal antibody (mAb) that binds to HER1 overexpressing tumor cells. Cetuximab has a direct effect on HER1-positive cancer cells, but it also can activate immune cells that bear receptors for the Fc (constant portion) of IgG such as natural killer (NK) cells. NK cells have an activating Fc receptor for IgG (FcγRIIIa), which mediates Ab dependent cellular cytotoxicity (ADCC) and enhances production of interferon-γ (IFN-γ) in response to Ab-coated targets. Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that stimulates IFN-γ production from NK cells. We hypothesized that IL-12 would enhance the anti-tumor activity of cetuximab by activating the FcR effector mechanisms of NK cells. METHODS: Expression of HER1 was measured on human papilloma virus (HPV)-positive (UD-SCC2, UM-SCC47) and HPV-negative (Cal27, UM-SCC74B) SCCHN cell lines by immunoblot analysis and flow cytometry. NK cells from normal donors were treated overnight with IL-2 (100 U), IL-12, IL-15, or IL-21 (all 10 ng/mL) and tested for ADCC versus cetuximab-coated cancer cells in a 4 hr (51)Cr assay. Release of cytokines by NK cells in response to cetuximab-coated cells was measured by ELISA. Phosphorylation of the ERK transcription factor in NK cells was measured by flow cytometry. The efficacy of combination therapy with cetuximab plus IL-12 was evaluated in a murine tumor model of head and neck cancer. RESULTS: All cell lines showed >99% expression of HER1 by flow cytometry and immunoblot analysis except UM-SCC74B (73%). Normal NK cells mediated 49.4% lysis of cetuximab-coated SCCHN cell lines as compared to 7.6% lysis of cells treated with control IgG (P = .0002). NK cell lysis of cetuximab-coated SCCHN cells was markedly enhanced by 12 hr pre-treatment of NK cells with IL-12 (71.6% lysis, P = .005 vs cetuximab alone). As a control, IL-12-activated NK cells were tested against IgG-treated cells. ADCC under these conditions was just 21.7%. Similar levels of lysis were noted for both HPV-positive and HPV-negative and cell lines. Other NK cell activating factors such as IL-2, IL-15, and IL-21 were also able to enhance NK cell ADCC. The stimulus of IL-12 and cetuximab-coated tumor cells induced the synergistic production of nanogram levels of IFN-γ (>6-fold increase over controls) (P < .001). A similar effect was seen for NK cell production of the chemokines RANTES, MIP-1α, and IL-8. Phosphorylation of ERK (which is critical for FcR-mediated ADCC and cytokine production) was enhanced in NK cells exposed to IL-12 and IgG as compared to control conditions. The combination of cetuximab plus IL-12 resulted in a reduction in tumor burden when compared to either agent alone in a murine xenograft model of SCCHN. CONCLUSION: Cytokine stimulation of NK cells in the presence of cetuximab-coated head and neck cancer cells leads to enhanced NK cell mediated ADCC and cytokine secretion independent of tumor cell HPV-status. Cytokine administration could be a useful adjuvant in the cetuximab treatment of HER1-positive head and neck cancer.

Monoclonal antibody therapy of pancreatic cancer with cetuximab: potential for immune modulation.

Pancreatic cancer is a devastating disease, with a median survival of around 6 months for patients with stage IV disease. The epidermal growth factor receptor (EGFR, or HER1) belongs to the erbB receptor tyrosine kinase family. HER1-mediated cell signaling has been shown to play a major role in promoting tumor proliferation, angiogenesis, metastasis, and evasion of apoptosis. Over-expression of HER1 is observed in multiple human malignancies, including colorectal, lung, breast and pancreatic cancers. In pancreatic carcinoma, over-expression of HER1 is observed in greater than 70% of patients and is associated with a poor prognosis and a significant decrease in survival. Cetuximab (Erbitux) is a chimeric monoclonal antibody (mAb) that binds to the extracellular domain of the HER1 molecule preventing ligand binding and promoting internalization and subsequent degradation of the HER1 receptor. Cetuximab has shown anti-tumor activity either alone or in combination with other agents and is currently FDA approved for use in both squamous cell carcinoma of the head and neck (SCCHN) and colorectal carcinoma. Research efforts continue to elucidate a possible role for cetuximab in the treatment of pancreatic cancer. Despite promising preclinical work, phase II and phase III clinical trials have failed to consistently show efficacy of cetuximab treatment in advanced pancreatic cancer either alone or in combination with cytotoxic agents. Alternative approaches to HER1 blockade and mAbs including immune modulation with cytokines might be necessary in order to improve the efficacy of mAbs in pancreatic cancer therapy.

Immune modulation with interleukin-21.

Interleukin 21 (IL-21) is produced by activated CD4(+) T cells. The IL-21R shares the common receptor gamma-chain with IL-2, IL-4, IL-7, IL-9, and IL-15, is widely expressed on immune cells, and mediates a variety of effects on the immune system. IL-21 enhances the proliferation, antigen-induced activation, clonal expansion, IFN-gamma production, and cytotoxicity of NK cells and T cells. The antitumor actions of IL-21 have been variously attributed to NK cell and CD8(+) T cell cytotoxicity, CD4(+) T cell help, NKT cells, and the antiangiogenic properties induced by IFN-gamma secretion. In clinical trials IL-21 has been well tolerated and induces a unique pattern of immune activation. IL-21 is therefore an excellent candidate for use in immune therapy.

TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems.

Mutations in TOO MANY MOUTHS (TMM), which encodes a receptor-like protein, cause stomatal patterning defects in Arabidopsis leaves but eliminate stomatal formation in stems. Stomatal development in wild-type and tmm stems was analyzed to define TMM function. Epidermal cells in young tmm stems underwent many asymmetric divisions characteristic of entry into the stomatal pathway. The resulting precursor cells, meristemoids, appropriately expressed cell fate markers such as pTMM:GFP. However, instead of progressing developmentally by forming a guard mother cell, the meristemoids arrested, dedifferentiated, and enlarged. Thus asymmetric divisions are necessary but not sufficient for stomatal formation in stems, and TMM promotes the fate and developmental progression of early precursor cells. Comparable developmental and mature stomatal phenotypes were also found in tmm hypocotyls and in the proximal flower stalk. TMM is also a positive regulator of meristemoid division in leaves suggesting that TMM generally promotes meristemoid activity. Our results are consistent with a model in which TMM interacts with other proteins to modulate precursor cell fate and progression in an organ and domain-specific manner. Finally, the consistent presence of a small number of dedifferentiated meristemoids in mature wild-type stems suggests that precursor cell arrest is a normal feature of Arabidopsis stem development.